The finding that individuals bearing genetic or acquired deficiency in C1q or C4 almost always develop lupus has been referred to as the Lupus Paradox. Like many loci within the Human Leukocyte Antigen locus (HLA), the complement C4 locus, which is located in the class III region, is highly polymorphic. There are two highly conserved isotypes, C4A and C4B, which encode structural variability in the isotypic region (4 amino acids) which regulate covalent binding of the internal thioester. While the most common haplotype is C4A and C4B, the number of alleles at the C4 locus can vary from 0 to 4. For example, allelic deficiency in C4A or C4B occurs at a frequency of about 4% and 1%, respectively. In contrast to humans, mice express a single functional C4 allele; the DNA sequence of the murine C4 gene in the isotypic region is a hybrid of human C4A and C4B. A key function of C4 is to bind covalently to its target following enzymatic cleavage. C4 is highly reactive once it is activated and studies using small molecule ligands in vitro determined that C4A retains its activity longer and primarily forms amide bonds; whereas, C4B forms ester bonds with the antigen surface. It is predicted that the in vitro chemical difference will have significant effects on C4 function in vivo. The availability of animal models to test the importanc of the two isoforms in vivo would be important to move the field forward. The goal of the current proposal is characterize two novel strains of mice in which the C4 locus was edited in the isotypic region to express either C4A or C4B protein. Aim 1: Characterize gene edited strains of mice bearing hu C4A or C4B isotypic sequences. We expect to learn from this aim if C4A and C4B differ in their protection of mice against viral and bacterial pathogens. Aim 2: Test the hypothesis that structural differences in the isotypic region of C4 influence protection in a murin model of lupus. We expect to learn from this aim if the C4A protein is more efficient than C4B in clearance of nucleolar antigens and in protection in a murine lupus model. Summary: The successful completion of this study will not only provide valuable reagents and novel tools to push the field forward but could lead to development of protein replacement therapy.